The separation of molecules in biological fluids is a basic procedure important in clinical diagnosis and disease treatment, and the most elemental device for solute separation is a porous membrane filter. Thus, a revolutionary advance in filter technology has the potential to impact many areas of human health. Typical filter materials are made as mats of plastic or cellulose polymers. Filters manufactured in this manner naturally contain a wide distribution of pore sizes and the smallest pores will eventually clog with small molecules of the filtrate. The abundance of small pores and large filter thicknesses are the two major sources of resistance to flow across membrane filters (Tong et al., “Silicon Nitride Nanosieve Membrane,” Nano Letters 4:283-287 (2004)).
Two types of nanofabricated membranes have been previously developed for molecular separations. In the first type, molecules pass through channels with nanoscale diameters and lengths between 6 μm and 60 μm. Channel-type filters have been assembled by impregnating mesoporous alumina or polycarbonate host membranes with silica (Yamaguchi et al., “Self-assembly Of A Silica-surfactant Nanocomposite In A Porous Alumina Membrane,” Nature Materials 3:337-341 (2004)) or gold (Lee et al., “Electromodulated Molecular Transport In Gold-Nanotubule Membranes,” J. Am. Chem. Soc. 124:11850-11851 (2002)), and by the selective removal of silicon dioxide (SiO2) from corrugated assemblies of silicon and polysilicon (Martin et al., J. Controlled Release 102:123-133 (2005)). Channel-type membranes have been used successfully to separate small ˜1 nm) molecules from proteins (˜5 nm) (Yamaguchi et al., “Self-assembly Of A Silica-surfactant Nanocomposite in a Porous. Alumina Membrane,” Nature Materials 3:337-341 (2004)), and to slow the diffusion of proteins for drug delivery applications (Martin et al., “Tailoring Width of Microfabricated Nanochannels To Solute Size Can Be Used To Control Diffusion Kinetics,” J. Controlled Release 102:123-133 (2005)). While the large thickness of channel-type membranes provides the mechanical stability needed for practical applications and large-scale separations, flow resistance (Tong et al., “Silicon Nitride Nanosieve Membrane,” Nano Letters 4:283-287 (2004)) and diffusion time increase directly with membrane thickness. Novel aspects of channel membranes, such as the sub-Fickian movement of molecules constrained to move in single file (Wei et al., “Single-file Diffusion of Colloids in One-Dimensional Channels,” Science 287:625-627 (2000)), can be expected to slow flux further. Long channels also create a lag between the initial introduction of a mixture and the appearance of a species at the backside of the membrane. For small macromolecules (˜1 nm) this delay can be several hours (Yamaguchi et al., “Self-assembly Of A Silica-surfactant Nanocomposite in a Porous Alumina Membrane,” Nature Materials 3:337-341 (2004)). Thus, while the use of channel-type nanomembranes for steady separations is possible, their use in rapid separation procedures seems unlikely.
The issue of transport efficiency is addressed by a second type of nanoporous membrane where the membrane is roughly as thick (˜10 nm) as the molecules being separated. As an array of molecularly sized holes in a molecularly-thin plane, this type of membrane achieves a structural limit that can help maximize filtration rates (Tong et al., “Silicon Nitride Nanosieve Membrane,” Nano Letters 4:283-287 (2004); Chao et al., “Composite Membranes From Photochemical Synthesis of Ultrathin Polymer Films,” Nature 352:50-52 (1991)). In published work, however, such an ultrathin membrane has been fabricated only by individually drilling 25 nm pores in a 10 nm thick silicon nitride membrane using an ion beam (Tong et al., “Silicon Nitride Nanosieve Membrane,” Nano Letters 4:283-287 (2004)). To provide mechanical integrity, this ultrathin membrane was suspended over 5 micron holes patterned in a much thicker underlying membrane. Large scale production of this type of membrane is impractical because the pore drilling procedure is expensive and extremely slow (˜20 hours/cm2) (Tong et al., “Silicon Nitride Nanosieve Membrane,” Nano Letters 4:283-287 (2004)). This method of manufacture would not lend itself to a commercially useful filtration membrane.
The implementation of microfluidics technology in small-scale protein separation and detection devices would benefit from the development of an electrically switchable nanofluidic filter. Such a filter would allow the construction of purification systems that can be electronically tuned to capture proteins with sizes specified by a user. In the case of a protein that is too dilute for detection or analysis, a dynamic filter could be programmed to first concentrate by trapping the protein as solute flows past, and then release the protein into an analysis chamber. There are several notable examples of switchable filters in the recent literature. In one example the charge on the membrane is actively manipulated to repel like-charged species from entrance into pores (Schmuhl et al., “SI-Compatible Ion Selective Oxide Interconnects with High Tunability,” Adv. Mater. 16:900-904 (2004); Martin et al., “Controlling Ion-Transport Selectivity in Gold Nanotubule Membranes,” Adv. Mater. 13:1351-1362 (2001)). In other examples, an electro-osmotic flow is established that drives solute charges past the membrane (Kuo et al., “Molecular Transport through Nanoporous Membranes,” Langmuir 17:6298-6303 (2001)). These filters were made by composite assembly procedures that would be costly to integrate into a mass production. The resulting filters are also thick, requiring species to pass through micron-long channels before emerging as filtrate. Large filter thicknesses are a major sources of resistance to flow-across membrane filters (Tong et al., “Silicon Nitride Nanosieve Membrane,” Nano Letters 4:283-287 (2004), and precludes the use of these designs in rapid separation applications.
High Throughput Screening (HTS) assays are often designed to identify small compounds with the ability to disrupt protein-protein interactions. Such assays are used, for example, when searching libraries for compounds that block enzyme activity by interfering with substrate binding or compounds that prevent docking between viral and cellular surfaces. These assays require the design of custom probes that change fluorescence, luminescence, or absorbance in response to the particular binding event being targeted. The need to design, synthesize, and validate custom probes for each drug strategy, explains why assay development remains a major bottleneck in the drug discovery pipeline (Inglese et al., 2007, Nat Chem Biol 3:438-441; Pollok, 2005, Nat Rev Drug Discov 4:1027).
The need to screen thousands of test compounds in drug discovery has driven the development of low-volume, high-density screening techniques involving 384, 1536 and 3456 multi-well plate formats. Automated plate readers and robotic liquid handling systems are designed to work with these plates to achieve high throughput. Due to manufacturing limitations however, the highest density plates containing transwell membranes have only 96 wells. The difficulty is that membranes are assembled into transwell devices by cutting them from macroscopic polymer sheets and then bonding with sonic welding, and these methods become increasingly difficult to control as well dimensions decrease at higher densities. Thus high throughput systems for nanoparticle screening at densities higher than 96 transwells will require new membrane technologies that are compatible with high-density formats.
The present invention is directed to overcoming these and other deficiencies in the art.